The accurate distribution of the replicated genome during cell division is crucial for genomic inheritance and cellular fitness. Conversely, disruptios in any of the steps associated with mitosis (the process in which a parental cell divides into two identical daughter cells) are often correlated with disease, especially cancer. The rapidly emerging picture is that mitosis is driven by the intricate balance between cell cycle kinases and phosphatases, especially the ser/thr phosphatase protein phosphatase 1 (PP1). While PP1 itself has a broad specificity, it acts in a highly specific manner by forming stable complexes (holoenzymes) with a host of regulatory proteins that direct its activity and localization. Our lon-term goal is to understand the structural and functional mechanisms that control PP1 activity in health and disease, an area in which our laboratory has made many fundamental contributions. Here we describe a complete research plan to understand the regulation of PP1 in the nucleus. The presented research project uses a powerful integrated approach that combines X-ray crystallography and NMR spectroscopy with biochemical and cell biology experiments to obtain novel insights into the molecular mechanisms that regulate PP1 activity during distinct stages of the cell cycle. Specifically, we are focusing on the regulation of PP1 by four PP1-targeting proteins: 1) Repoman (recruits PP1 onto mitotic chromatin at anaphase), 2) PNUTS (PP1 nuclear targeting subunit), 3) Knl1 (Kinetochore null protein 1) and 4) the ASPP (Apoptosis-stimulating of p53 protein) family of proteins. Abnormal expression of each of these regulators is associated with the increased incidence and severity of multiple cancers. Detailed descriptions of the molecular interactions of these regulators with PP1, which are currently missing, are needed for a comprehensive functional understanding of these important holoenzymes. In our combined efforts, we will: 1) determine the structures of the free form of these PP1 regulators, 2) determine the structures of the PP1 holoenzymes and 3) determine how these complexes direct and regulate PP1 activity. We will then leverage these protein and protein complex structures to elucidate, at a molecular level, the biological functions and modes of action of these key nuclear PP1 holoenzymes. The research described in this proposal leverages the extensive expertise of leading investigators in the PP1 research field, as well as takes advantage of the best possible national and international collaborators. Finally, it has the preliminary data that demonstrates that this work will provide unique, novel insights into the molecular regulation of PP1 and its fundamental roles during distinct stages of the cell cycle.